Revised sequence of the tetracycline-resistance gene of pBR322

&VW. 22 (19X3) 277 -280

217

Elsewer

Science

Publishers

Short

Communications

Revised

sequence

(Plasmid

vector;

of the tetracycline-resistance

open reading

frame;

predicted

gene of pBR322 polypeptide;

maxicell

system)

Keith W.C. Peden Howard Hughes Medical Institute Laboratory,, Departmettt of Moleculur Biolog, und Genetics, Johns Hopkms School of Medicine, Baltimore, MD 21205 (U.S.A.) Tel. (301) Y55-3652 (Received

December

(Accepted

January

Umcersrt~

3 1 st. 1982) 10th.

1983)

SUMMARY

A revised sequence of the tetracycline-resistance gene of pBR322 is reported. The change, the presence of an additional CG base pair at position 526, adjusts the published sequence to allow an open reading frame from nucleotides 8661273 (new number) and increases the size of the plasmid to 4363 bp. The predicted polypeptide encoded by this region would contain 396 amino acid residues and have a calculated M, of 41518. A polypeptide of the predicted size has been reported previously when pBR322 is used as template

in the maxicell

system.

Tetracycline resistance in bacteria is thought to be mediated by a membrane-associated protein

Data

on the location

tance gene within

of the tetracycline-resis-

pBR322

have come from inser-

which acts to exclude tetracycline from the cell (for review, see Chopra and Howe, 1978). The elucidation of the mechanism of resistance will

tion mutational analysis and deletion analysis. Insertion of DNA into the C/u1 site (nucleotide 23),

depend upon the determination and characterization of the protein(s) involved. With the publication of the sequence of pBR322 (Sutcliffe, 1979) the sequence of a protein conferring tetracycline resistance should be evident. However, there is a discrepancy between the seven polypeptides that theoretically could be produced from the region involved with tetracycline resistance, and the single polypeptide of M, about 40000 actually found (Sancar et al., 1979).

tide 375) SphI site (nucleotide 562), Sal1 site (nucleotide 651) or XmaIII site (nucleotide 939) causes loss of tetracycline resistance (e.g., Bolivar et al., 1977). Deletions extending from the PuuII site (2066) to nucleotide 1285 do not inactivate tetracycline resistance, whereas those that remove the 7’aqI site at 1267 do (K.W.C.P., unpublished results). These findings demonstrate that the region extending at least from nucleotide 23 to 1267 is involved in determining tetracycline resistance. A modification reported here to the published sequence of pBR322 (Sutcliffe, 1979) establishes an open reading frame from nucleotides 8661273,

Abbreviation:

bp. base

037x- 1 I 19/83/$03.00

pairs.

( 1 1983 Elsevier

Science

Publishers

Hind111 site (nucleotide

29). BarnHI

site (nucleo-

77x

aud thus a single polypeptide

could span the entire

region attributed to tetracycline resistance. The sources of pBR322 DNA (Bolivar et al., 1977) used

in this work

Hamilton

0.

were from

Smith

and

the laboratory

prepared

of

as described

previously (Peden et al., 1982). and from the Weizmann Institute of Science (gift of Chaim Kahana). The

nuclcotide

nucleotides

sequence

pBR322

between

375 and 651 was determined

for both

strands

usmg the method

(1980).

Labelling

of

of Maxam

at the BanrHI

site) was accomplished

with

and Gilbert

site (or the SulI

DNA

I

polymerase

c

from ~1rc~r~~c~occu.s luteu.r and the appropriately labelled denxynucleoside triphosphate. After labelling. a second digestion with S&I (after labelling the BunrHI site) or Bur?zHl (after labelling at the

C

Sri/I site) was carried out to produce fragments lahelled at one end. Labelling at the Sphl site was done

using

terminal

cordycepin-5’-triphosphate and a second digestion

transferase

and

[a-”

PI-

(Tu and Cohen. 1980). with BtrmHI produced a

fragment labelled at one end. Fig. 1 shows the region of the sequencing gel between nucleotides 511 and 550 for one strand. At the position indicated four cytosine residues are present, whereas only three residues were reported previously (Sutcliffe. 1979). This change was confirmed both by sequencing the other strand and by sequencing from the .‘?,&I site. This finding was confirmed by sequencing the second isolate of pBR322. The addition of a single nucleotide pair to the sequence causes a change in the reading frames

such that the predicted

polypeptide

of 179

amino acids now becomes part of the predicted polypeptide of 338 amino acids. This new polypeptide contains 396 amino acids starting at the ATG (nucleotides 86688) and terminating at the TGA (nucleotides 127441276). Both its nucleotide and amino acid sequences are presented in Fig. 2. and its calculated M, is 41 5 18. As pointed out by Sutcliffe (1979). the amino terminal portion of the 179 amino acid polypeptide shows homology with the corresponding region of the /3-lactamase protein, both having a high proportion of hydrophobic residues. This is consistent with the membrane association of these proteins. Insertion of DNA into the Hind111 site usually inactivates tetracycline resistance (Bolivar et al..

Fig. I. Sequencing between with ‘k/I using

51

This fragment

polymerase generated

a 276.bp

fragment

to sequence

(1980). the cleavage

an 8% polyacrylamlde gel exposed

by incorporation

of [u’~PJTTP

I from M~~~rowccus lurruc.

was subjected

and tiilbert

gene of pBK327

I and 550. Plasmid DNA was digested

and 3’ end labelled

DNA

with BarnHI Maxam

of the tetracycline-resistance

nucleotldes

gel (Sanger

labelled

Digestion at one end.

analysis

according

to

products

separated

on

and Coulson.

197X). and the

to autoradiography.

1977). The sequence in this region is a promoter by analogy with known promoters (Bolivar et al.. 1977) and presumably is the one used for expression of the tetracycline-resistance gene (Sutcliffe, 1979) being situated close (- 59) to the ATG of the 396 amino acid polypeptide (nucleotide 86). In summary. the revised nucleotide sequence of the gene specifying tetracycline resistance permits a single open reading frame from nucleotides 86-1273, and the predicted polypeptide of 396 amino acids would accommodate the genetic and biochemical activities attributed to this region. The addition of the single CG base pair increases the size of pBR322 to 4363 nucleotide pairs.

ACKNOWLEDGEMENTS

1 thank Daniel Nathans, Daniel Linzer for comments

Phoebe Mounts and on the manuscript,

L,

Ile

ATC

GTC

ACC

GCG

ATT

TAT

GCC

GCC

TCG

GCG

AGC

ACA

TGG

AAC

Val Thr Ala Ile Tyr Ala Ala Ser Ala Ser Thr Trp Am

GGG

TTG

GCA

TGG

ATT

GTA

GGC

GCC

GCC

CTA

TAC

CTT

GTC

WC

CTC

CCC

GCG

Gly Leu Ala Trp Ile Val Gly Ala Ala Leu Tyr LOU Val QJS LOU Pro ala

526.

-

/^

-

i

-

7.

<.”

,

>a-

“-^

^,,

--

“I_

Fig. 2. Nucleotide and amino acid sequences of the tetracycline resistance gene of pBR322. Nucleotide numbers refer to those of Sutcliffe (1979) with the addition of one after nucleotide

1238 TTG CGT CGC GGT GCA TGG AGC CGG GCC ACC TCG ACC TGA 1276 Leu Arg Arg Gly Ala Trp Ser Arg Ala Thr Ser Thr *

Leu

1142 CTG

1046 CAG GCC ATC CTG TCC AGG CAC CTA GAT CAC GAC CAT CAG CGA CAG CTT CAA GGA TCG CTC CCC CCT CTT ACC AGC CTA ACT TCG ATC ACT CGA CCG Gin Ala Met Leu Ser Arg Gin Val Asp Asp Asp His Gin Gly Gin Leu Gin Gly Ser Leu Ala Ala Leu Thr Ser Leu Thr Ser Ile Thr Cly Pro

950 CTG GGC TAC CTC TTG CTG GCG l-K GCG ACG CGA CXX TGG ATG CCC l-K CCC ATT ATG ATT C-l-TCTC GCT XC CCC GGC ATC GGG ATG CCC GCG lTG Leu Gly Tyr Val Leu Leu Ala Phe Ala Thr Arg Gly Trp Met Ala Phe Pro Ile Met Ile Leu ku Ala Ser Gly Gly Ile Gly Met Pro Ala Leu

854 ATC TTG CAC GCC CTC GCT CAA GCC l-E GTC ACT 'XT CCC GCC ACC AAA CGT TX GGC GAG AAG CAG GCC ATT ATC KC GGC ATC GCG GCC GAC GCG Ile Leu His Ala Leu Ala Gin Ala Phe Val Thr Gly Pro Ala Thr Lys Arg Phe Gly Glu Lys Gin Ala Ile Ile Ala Gly Met Ala Ala Asp Ala

758 GTA GGA CAG GTG CCG GCA GCG CTC TGG GTC ATT TTC GCC GAG GAC CGC TIT CGC TGG AGC GCG ACG ATG ATC GGC CTG TCG CTT GCG GTA TTC CGA Val Gly Gin Val Pro Ala Ala Leu Trp Val Ile Phe Cly Clu Asp Arg Phe Arg Trp Ser Ala Thr Met Ile Gly Leu Ser Leu Ala Val Phe Gly

662 CCC TTG AGA GCC TTC AAC CCA GTC AGC TCC TTC CGG TGG GCG CGG GGC ATG ACT ATC GTC GCC GCA CTT ATG ACT GTC 'IX TTT ATC ATG CAA CTC Pro Leu At-g Ala Phe Asn Pro Val Ser Ser Phe Arg Trp Ala Arg Cly Met Thr Ile Val Ala Ala Leu Met Thr Val Phe Phe Ile Met Gin Leu

566 GCA CCA TTC CTT GCG GCG GCG GTG CTC AAC GGC CTC AAC CTA CTA CTG GGC TCC TTC CTA ATG CAG GAG TCG CAT AAG CGA GAG CGT CGA CCG ATG Ala Pro Phe Leu Ala Ala Ala Val Leu Asn Gly Leu Asn Leu Leu Leu Gly Cys Phe Leu Met Gin Glu Ser His Lys Gly Glu At-g Arg Pro Met

470 CGG GCT CGC CAC TTC GGG CTC ATG AGC GCT TGT 'l-l-C GGC CTG GGT ATG GTG GCA GGC CCC CTG GCC GCG GGA C-K TN GGC GCC ATC TCC 'I-l-G CAT Arg Ala Arg His Phe Gly Leu Met Ser Ala Cys Phe Gly Val Gly Met Val Ala Gly Pro Val Ala Gly Gly IEU Leu Gly Ala Ile Ser Leu His

374 TGG ATC CTC TAC GCC GGA CGC ATC GTG GCC GGC ATC ACC GGC GCC ACA GGT GCG GTT GCT GGC GCC TAT ATC GCC GAC ATC ACC GAT GGG GAA GAT Trp Ile Leu Tyr Ala Gly Arg Ile Val Ala Gly Ile Thr Gly Ala Thr Gly Ala Val Ala Cly Ala Tyr IIe Ala Asp Ile Thr Asp Gly Glu Asp

278 GCA CTG TCC GAC CGC TTT GGC CGC CGC CCA GTC CTG CTC GCT TCG CTA CTT GCA GCC ACT ATC GAC TAC GCG ATC ATG GCC ACC ACA CCC GTC CTG Ala Leu Ser Asp erg Phe Gly Arg Arg Pro Val Leu Leu Ala Ser Leu Leu Gly Ala Thr Ile Asp Tyr Ala Ile Met Ala Thr Thr Pro Val Leu

182 CGG GAT ATC GTC CAT TCC GAC AGC ATC CCC AGT CAC TAT GGC GTG CTG CTA GCG CTA TAT GCG lTG ATG CAA TTT CTA 'ICC GCA CCC GTT (XC GGA Are Asp Ile Val His Ser Asp Ser Ile Ala Ser His Tyr Gly Val Leu Leu Ala Leu Tyr Ala Leu Net Cln Phe Leu Cys Ala Pro Val LeU Gly

86 ATG AAA TCT AAC AAT GCG CTC ATC GTC ATC CTC GGC ACC GTC ACC CTC CAT GCT GTA CXX ATA C&C 'I-I-G CTT ATG CCC GTA CTG CCG (XX (XC 'Tl'G MET Lys Ser Am Asn Ala Leu Ile Val Ile Leu Gly Thr Val Thr Leu Asp Ala Val Gly Ile Gly IEU Val Met Pro Val Leu Pro Gly LeU Ix%

280

Bill Pearson

for assistance

with the computer

anal-

ysis and Doris Wiczulis for typing the manuscript. This work was supported by Grant 5 PO1 CA165 19 from the National Cancer Institute.

Moldave.

K. (Eds.),

Academic

Press, New York.

Peden. K., Mounts, mammalian genomes

detected probe.

A., Hack.

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